Locus-specific chromatin proteomics using dCas-guided proximity labelling in aspergillus nidulans.

Abstract

Proximity labelling that uses promiscuous biotin ligases (BirA) fused to a bait protein is a powerful tool to identify protein interaction partners in vivo under different metabolic or developmental conditions. BirA can also be used to determine protein composition and interaction partners at specific chromatin locations when it is fused with enzymatically-disabled Cas9 (dCas9) and then guided to the location of interest by sgRNAs. We adapted this method (called CasID) for fungal cells using the nitrate assimilation gene cluster of A. nidulans as a model locus and estrogen-inducible expression of the dCas9-BirA fusion to improve condition-specific labelling. For method establishment, we first verified the presence of dCas-BirA and a known transcription factor at the nitrate locus by chromatin immunoprecipitation (ChIP). Results show that both dCas-BirA and the AreA transcription factor are present at the locus of interest under the conditions used for biotinylation. We then optimized the CasID procedure for efficient labelling and background reduction using the CasID-sgRNA strain and two control strains, one lacking the sgRNA and another one lacking the whole CasID system. Here we provide proof-of-concept for the suitability of the method by showing that biotinylated proteins are enriched in the CasID strains in comparison to the controls. After background reduction, 32 proteins remained in two independent experiments exclusively enriched in the Cas-ID-sgRNA strain. Among these proteins was NmrA, an AreA-interacting regulator, and we also found several chromatin-associated proteins. Overall, our results demonstrate that CasID is suitable for locus-specific labelling and identification of chromatin-associated proteins and transcription factors in A. nidulans. However, the high background of proteins that are biotinylated out of chromatin context or unspecifically attach to the affinity purification matrix needs to be addressed by implementing a set of rigorous controls. In summary, we herewith provide a detailed protocol for application of the method that proved to be useful for the identification of novel chromatin-associated proteins and their interaction partners at a specific genomic locus in divers metabolic and developmental conditions. AUTHOR SUMMARY: This study demonstrates that locus-specific proteomics can be carried out by dCas-BirA guided proximity labelling in Aspergillus nidulans. For establishment, we targeted the well-described bidirectional promoter region between niaD, a nitrate reductase, and niiA, a nitrite reductase. At this locus we could test by chromatin immunoprecipitation (ChIP) in combination with qPCR if both, the dCas9-BirA fusion as well as a central transcription factor are at the locus under the conditions of our CasID experiment. After this first control step, we considered that unspecific labelling by dCas-BirA during the time from translation to landing at the targeted chromatin locus may be one of the most relevant drawbacks of the method. Therefore, we developed a number of control strains that would allow us to clearly discriminate between background and sgRNA-dependent specific labelling at the locus. Our protein MS results validated these estimates and only considering the results of these controls enabled us to distinguish the set of locus-specific proteins from a very high general background. Finally, enrichment of biotinylated proteins through affinity purification with streptavidin resin and subsequent LC-MS/MS analysis showed that more than 800 proteins were detected in each sample, emphasizing the high background of the purification method. After background reduction of the control samples, we were able to identify 32 proteins which were exclusively detected in the test strain in two independent measurements, including several chromatin-associated proteins and NmrA, a negative regulator of the nitrate locus transcription factor AreA.